PROJECT SUMMARY The objectives of this research program are to understand the molecular mechanisms of ion permeation, ion selectivity, and gating in eukaryotic ion channels that generate or respond to intracellular calcium signals. The ion channels under study include the calcium release-activated calcium (CRAC) channel Orai, the calcium- activated chloride channel bestrophin (BEST), and the mitochondrial calcium uniporter (MCU). The channels play vital roles in cellular physiology and are tightly regulated. CRAC channels are necessary for the activation of immune response genes in T cells; mutations cause severe combined immunodeficiency-like disorders. BEST channels form anion-selective pores that are regulated by changes in the intracellular calcium concentration, by phosphorylation, and by changes in cell volume. Mutations in BEST channels cause inherited retinal degenerative diseases (bestrophinopathies) including a juvenile-onset form of retinal degeneration (Best vitelliform macular dystrophy). MCU is the primary means for calcium entry into mitochondria. Mitochondrial calcium uptake by MCU regulates ATP production, shapes cytosolic calcium signals, and controls a mitochondrial permeability transition that leads to cell death. We combine approaches to determine three-dimensional structures (X-ray crystallography and cryo-electron microscopy) with functional analyses (electrophysiology and fluorescence-based approaches) to dissect the molecular mechanisms of these ion channels. Our accomplished structural and functional studies of these channels reveal that each has a distinct architecture in comparison to other ion channels and regulates ion permeation, ion selectivity and gating in unique ways. For the CRAC channel Orai, the current aims are to discern how the channel transitions between closed and open states, how the binding of STIM, the channel's activator, drives this process, how the channel exquisitely discriminates calcium from other ions, and how the channel catalyzes ion permeation without overwhelming the cell with calcium. For BEST channels, we aim to capture structures that represent different gating states of the channel and discern the functional and molecular bases for calcium- dependent activation, inactivation, and anion selectivity. Further, we seek to discern the molecular and functional differences among mammalian BEST1-4 isoforms. Regarding MCU, we aim to study three- dimensional structure, investigate how the channel is regulated by calcium and protein-protein interactions, and probe the molecular basis of ion selectivity. The proposed studies will reveal principles of CRAC, BEST, and MCU channel function, thereby making significant contributions to our understandings of ion channels and the physiological processes they control.